Sybr green pcr kit

Total RNA extraction and data processing were conducted as described previously[41 (link)]. Briefly, the wild-type strain and dr0997 mutant strain were cultured until the OD₆₀₀ reached 0.4, and then harvested. Total RNA was extracted using the Whole RNA Extraction kit (Promega, Madison, WI USA). RNA quality and quantity were evaluated by measuring the A₂₆₀/A₂₈₀ ratio with a NanoDrop-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and Denaturing Agarose Gel Electrophoresis.
The qRT-PCR assay utilized RNA samples that obtained under different conditions. The wild-type and mutant strains were grown in TGY until the OD₆₀₀ reached 0.5. Each culture was divided in two halves: one half of the culture was treated with H₂O₂ at a final concentration of 50 mM, while the other half was used as the non-treated control. Total RNA was extracted as mentioned previously. First-strand cDNA synthesis was conducted in 20-μl reactions containing 1 μg of purified RNA and 3 mg of random hexamers. The SYBR Green PCR kit (Tiangen, Beijing, China) was used for PCR amplification according the manufacturer’s instructions, and all assays were performed using the Mx3005P^TM Real-time Detection System (Stratagene, La Jolla, CA, USA).

TRI Reagent (catalogue no. T9424; Sigma, Germany), miRNeasy Micro Kit (cat alogue no. T9424 217084; Qiagen, Germany), RNase-Free DNase Set (cat alogue no. 79254; Qiagen), Tiangen miRcute miRNA cDNA First-Strand Synthesis Kit (cat alogue no. KR201; Tiangen, Beijing, China), SYBR Green PCR kit (cat alogue no. Fp411-02; Tiangen, Beijing, China), Agilent Bioanalyzer 2100 analyzer (Agilent Technologies, Santa Clara, CA, USA); Scanner G2505C (Agilent Technologies), feature extraction (version 10.7.1.1, Agilent Technologies), data processing software Genespring (version 13.1, Agilent Technologies), and Affymetrix Agilent Human miRNA, Release 21.0 (8 × 60K, Design ID: 070156) chip were utilized for this study.

Primer sequences were designed based on GenBank sequences with Primer Premier 5.0 software (Table 1). The PCR primers were synthesized by Shanghai Jima Pharmaceutical Technology (Shanghai, China). mRNA was extracted using TRIzol (Thermo Scientific), and RNA was reverse transcribed with a TIANScript RT Kit (Tiangen Biotechnology, Beijing, China) according to the manufacturer's instructions. The reaction system was prepared with a SYBR Green PCR kit (Tiangen Biotechnology), and mRNA amplification and quantification were performed using an ABI 7500 fluorescence quantitative PCR instrument (Applied Biosystems, Foster, USA) according to the instructions. The reaction procedure was as follows: preincubation at 95°C for 5 min, amplification at 95°C for 10 s and 60°C for 60 s for 40 cycles, and separation at 95°C for 10 s and 60°C for 60 s. Target gene expression was normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and relative expression was determined using the 2^−ΔΔct method.